Genomic characterization of Campylobacter isolates in Huzhou, China

Campylobacter is a major foodborne pathogen that causes outbreaks and sporadic gastrointestinal disease, creating a serious disease burden. Campylobacter strains isolated from diarrhea cases (n = 11) and raw poultry meat products (n = 2) in Huzhou, including 11 Campylobacter jejuni and 2 Campylobacter coli strains, were subjected to virulence gene, drug resistance gene, genetic correlation, antibiotic resistance, and multilocus sequence typing (MLST) analyses. The 13 Campylobacter isolates were divided into 12 sequence types (STs), one of which was a new ST. The isolated strains contain multiple virulence-related genes. Drug sensitivity analysis showed that the resistance rate of the 13 isolates to nalidixic acid, ciprofloxacin, and tetracycline was 92.3%. Genome sequencing indicated that all 11 strains of C. jejuni carried the tet(O) and blaOXA resistance genes, and 2 strains of C. coli carried multiple drug resistance genes. Phylogenetic analysis based on core-genome single-nucleotide polymorphisms indicated that the 11 C. jejuni isolates from diarrhea patients and food sources are not closely phylogenetically related.


Introduction
Campylobacter is a zoonotic human pathogen that can cause diarrhea [1], and the main Campylobacter species causing infections in humans are C. jejuni and C. coli (accounting for more than 90%) [2]. The main clinical symptoms of Campylobacter infection are diarrhea and fever, and immune damage such as Guillain-Barre syndrome (GBS) can also occur [3]. Campylobacter is the most commonly reported foodborne pathogen causing outbreaks and sporadic gastrointestinal illness in developed countries and thus carries a significant disease burden [4], with up to 2.5 million cases in the United States each year [5]. Campylobacter causes more diarrhea than Salmonella or Shigella in developing countries [6,7]. Sporadic diarrhea cases [8] and food-borne illnesses [9] caused by Campylobacter-contaminated foods have been frequently reported in China in recent years. Currently, the pathogenic mechanism of Campylobacter infection in humans is unclear [10]. Campylobacter mainly infects the body through adhesion, colonization, invasion, toxin production, and other mechanisms, leading to disease; during this process, a variety of virulence genes participate in the expression of virulence factors [11]. The flagellin gene flaA is closely related to the invasiveness and pathogenicity of bacteria, while the CadF gene encodes an outer membrane protein of Campylobacter jejuni that plays an important role in the adhesion and invasion process between C. jejuni [12]. The cdt gene cluster is composed of cdtA, cdtB, and cdtC in series, among which cdtB occurs widely and is correlated with cytolethal distending toxin titer [13].
The presence of various virulence genes is common in cases of campylobacteriosis. Drug resistance is a major problem affecting Campylobacter research and infection control. In 2013, fluoroquinolone-resistant Campylobacter and macrolide-resistant Campylobacter were listed as drug resistance threats affecting public health by the United States Centers for Disease Control and Prevention [14]. In China, drug resistance in Campylobacter has been reported; in particular, multi-drug resistant strains of C. jejuni have emerged as a public health concern [7].
Conventional methods for the isolation and identification of Campylobacter include enrichment culturing, selective isolation, and biochemical identification. The culture conditions for Campylobacter are harsh and cultivation time is long, which prevents the rapid diagnosis of food-borne diseases caused by Campylobacter infection. In recent years, whole-genome sequencing (WGS) has been widely employed in Campylobacter research [15]. Through systematic analysis of WGS data, the genomic characteristics and evolutionary relationships of various Campylobacter species can be determined. In this study, the whole genomes of 11 strains of C. jejuni and 2 strains of Campylobacter coli isolated from diarrhea patients and food sources were sequenced to obtain preliminary information about the genotypes and distribution characteristics of drug resistance genes, virulence genes, as well as the phylogenetic and evolutionary relationships among strains isolated in Huzhou. This study provides technical support for food safety risk assessment, prevention, and control of campylobacteriosis occurrence and development in the Huzhou area.

Ethics statement
This study was approved by the human research ethics committee of the Huzhou Center for Disease Control and Prevention. The only human material used in this study is stool samples taken from patients for routine assessment. Oral informed consent was obtained from each participant.

Bacterial isolates
A total of 13 strains of Campylobacter were isolated from diarrhea patients (stool samples) and raw poultry meat products collected at food markets during foodborne disease surveillance in 2022 across several regions of Huzhou. The 11 strains of Campylobacter obtained from diarrhea patients included 9 strains of C. jejuni and 2 strains of C. coli. Both strains of Campylobacter isolated from raw poultry meat products were C. jejuni. The 13 isolates were stored at −80˚C in porcelain culture storage tubes (Qingdao Haibo, China).

Antimicrobial susceptibility testing
The antimicrobial susceptibility of the 13 Campylobacter isolates was tested using the agar dilution method. The 11 antibacterial agents tested included the macrolide (erythromycin and azithromycin), quinolone and fluoroquinolone (nalididic acid and ciprofloxacin), aminoglycoside (gentamicin and streptomycin), chloramphenicol (chloramphenicol and florfenicol), tetracycline (tetracycline), ketolactone (telimycin), and lincosamide (clindamycin) classes of antibiotics. Antimicrobial susceptibility was interpreted as sensitive, intermediate, or resistant, with reference to the interpretation criteria described in the National Antimicrobial Resistance Monitoring System Drug Sensitivity Test Guidelines. The quality control strain for antibiotic resistance testing, C. jejuni (ATCC 33560), was obtained from Qingdao Zhongchuang, China.

Genome sequencing
The whole-genome DNA libraries of 13 Campylobacter strains were constructed used the NGSmaster pathogen metagenomic one-stop library and kit. Next, the whole genome was sequenced using a NextSeq 550 sequencer (Illumina, USA).

Multilocus sequence typing (MLST)
The sequences of seven housekeeping genes (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) in the genome were used to determine the sequence type (ST) of C. jejuni and C. coli isolates through comparison with pubMLST (https://pubmlst.org/). If no ST is listed in the database matching the sequence of the strain, sequence alignment was conducted using BLAST to determine the new allele number of the housekeeping gene, which was applied as a new ST.

Analysis of virulence genes
The virulence gene distribution was analyzed through WGS analysis of the strain. The genomes of C. jejuni and C. coli were submitted to The Virulence Factor Database (VFDB) to obtain virulence gene profiles. Pheatmap software was used to draw heatmaps based on the presence or absence of virulence genes.

Analysis of drug resistance genes
The genomes of C. jejuni and C. coli were submitted to CARD (Comprehensive Antibiotic Resistance Database), and ABRicate software was used to predict and analyze the antibiotic resistance genes of the sequenced strains.

Genetic correlation analysis
Snippy, Gubbins, and other software were used to sort the sequencing results and obtain the core-genome single-nucleotide polymorphisms (SNPs) of the strains. Sequence alignment and homology analysis of the 13 Campylobacter isolates were performed with Raxmal software.

Genome sequencing
The average length of the genome sequences of 11 C. jejuni isolates was 172.15 kbp, and the GC content was 30%. The average length of the genomes of C. coli isolates was 168.97 kbp, and GC content was 31%. These lengths and GC contents are consistent with the genomic characteristics of C. jejuni and C. coli (Table 1).

MLST molecular typing
The 11 strains of C. jejuni were divided into 10 STs, with one strain (strain CJS210762) belonging to a new ST (ST, 11775). The two strains of C. coli were associated with two STs ( Table 1).

Analysis of virulence genes
Analysis of virulence genes in the genomes of 13 Campylobacter strains showed that most strains carried more than 70 different virulence-related genes. No significant aggregation in the distribution of virulence genes was found among strains. Differences in the distribution of virulence genes were observed between C. jejuni and C. coli, as well as among strains of a given species. All 11 strains of C. jejuni contained three cytotoxic genes, cdtA, cdtB, and cdtC (Fig 1).

Antimicrobial resistance and drug resistance gene analyses
The resistance rate of the 11 strains of C. jejuni to quinolones and tetracycline was 100%, and the resistance rate of the 2 strains of C. coli to tetracycline was 100%. Twelve of the 13 Campylobacter strains were resistant to three or more antibacterial drugs, with the remaining C. coli strain resistant to just one bacterial drug. Analysis of drug resistance genes indicated that the number of drug resistance genes was higher for C. coli than C. jejuni. All 11 strains of C. jejuni had tet(O) resistance genes and β-lactam antibiotic (blaOXA) resistance genes. Mutation analysis of the quinolone antibiotic resistance determinant region showed that C257T mutation occurred in the gyrA genes of 12 strains, all except CJS22016, which may be related to the quinolone antibiotic resistance of that strain (Table 2).

Genetic correlation analysis
Core-genome SNP (cgSNP) clustering analysis based on the genome sequences of 13 isolates showed that C. jejuni and C. coli fall into two distinct groups (Fig 2). Using the genome  sequence of NCTC 11168, the standard strain of C. jejuni, as the reference strain, cluster analysis of the genomes of 11 strains of C. jejuni indicated no apparent clustering of strains from different sources (Fig 3).

Discussion
Campylobacter infection is considered a major cause of bacterial diarrhea in both developed and developing countries and is therefore an important public health problem [16]. Surveillance of Campylobacter in patients with diarrhea has been conducted in many provinces  and cities in China, and Campylobacter has a higher detection rate than Salmonella, Shigella, and diarrhea-causing Escherichia coli [17][18][19]. With the improvement of living standards, food consumption is increasingly diversified and products are consumed in large quantities. The safety hazards associated with Campylobacter are also increasingly prominent. Therefore, clarifying the genetic characteristics of Campylobacter in this region provides technical information to support effective prevention of the ongoing Campylobacter epidemic. WGS can be used to elucidate the molecular characteristics of virulence and drug resistance in various pathogens at the molecular level. In this study, WGS data for 9 strains of C. jejuni and 2 strains of C. coli isolated from stool samples of diarrhea patients as well as 2 strains of C. jejuni isolated from raw poultry meat samples were analyzed. The genomic characteristics of C. jejuni from these two sources were investigated, and no significant clustering was found. MLST is a classical bacterial molecular typing technique widely used to identify relationships among bacterial clones [20]. In this study, MLST analysis of 11 strains of C. jejuni and 2 strains of C. coli demonstrated the genetic diversity of their genotypes, in accordance with previous reports [17,19,21]. One C. jejuni strain was found to represent a new ST (ST11775). ST-464 is the most common ST in China [17,19], and was detected in this study.
Bacterial adhesion, invasion, and flagellar activity are related to virulence and pathogenicity. Previous studies have confirmed that virulence genes such as flaA, flaB, CadF, and cdt are prevalent in C. jejuni and C. coli [22]. In this study, 13 Campylobacter isolates from two major sources carried more than 70 virulence-related genes, and the proportions of strains carrying the fibril binding protein gene cadF, invasion-related virulence gene ciaB, and flagellar gene flaA were very high. All 11 strains of C. jejuni contained three cytotoxic genes, cdtA, cdtB, and cdtC. This finding is consistent with Chinese and international reports [23,24].
In recent years, fluoroquinolone and tetracycline antibiotics have been used as growth promoters in animal husbandry, resulting in a substantial increase in the proportion of drug-resistant bacteria [25]. The main mechanism of quinolone resistance is mutation of the GyrA gene in C. jejuni, while tetracycline resistance in Campylobacter is mainly due to the ribosome protective protein encoded by the tet(O) gene [26]. The results of drug sensitivity analysis showed that all 13 Campylobacter isolates were highly resistant to tetracycline and fluoroquinolone antibiotics. Drug resistance gene analysis indicated that all 11 strains of C. jejuni and 2 strains of C. coli had mutations of the tet(O) drug resistance gene and gyrA gene, with the 2 strains of C. coli carrying significantly more drug resistance genes than C. jejuni. However, this analysis also showed that the drug resistance genes were not fully expressed in the phenotypes of the isolated strains. The study of Campylobacter genetic characteristics has been suggested as an important means to elucidate the epidemic characteristics and evolutionary relationships of Campylobacter. In this study, based on cgSNP clustering analysis of core genes of the sequenced strains, C. jejuni and C. coli were found to cluster into two groups. The genomes of 11 C. jejuni isolates with different sources showed high diversity.

Conclusion
We analyzed the antimicrobial resistance genes and conducted WGS of Campylobacter strains isolated from diarrhea cases and raw poultry meat in Huzhou. We assessed antibiotic resistance in Campylobacter in that region as well as the genetic characteristics of Campylobacter strains. This information will lay a foundation for the identification and pathogenicity analysis of sporadic and clustered outbreaks of Campylobacter infections in Huzhou.